This invention relates to certain compounds, and their pharmaceutically acceptable salts, which inhibit matrix metalloproteases (MMPs), particularly MMP-3, MMP-12 and MMP-13. They are therefore useful in the treatment of mammals having conditions alleviable by inhibition of MMPs, especially MMP-3, MMP-12 and MMP-13.
MMPs constitute a family of structurally similar zinc-containing metalloproteases, which are involved in the remodelling, repair and degradation of extracellular matrix proteins, both as part of normal physiological processes and in pathological conditions. Since they have high destructive potential, the MMPs are usually under close regulation, and failure to maintain MMP regulation has been implicated as a component of a number of conditions. Examples of conditions where MMPs are thought to be important are those involving bone restructuring, embryo implantation in the uterus, infiltration of immune cells into inflammatory sites, ovulation, spermatogenesis, tissue remodelling during wound repair and organ differentiation such as such as in venous and diabetic ulcers, pressure sores, colon ulcers for example ulcerative colitis and Crohn""s disease, duodenal ulcers, fibrosis, local invasion of tumours into adjacent areas, metastatic spread of tumour cells from primary to secondary sites, and tissue destruction in arthritis, skin disorders such as dystrophic epidermolysis bulosa, dermatitis herpetiformis, or conditions caused by or complicated by embolic phenomena, such as chronic or acute cardiac or cerebral infarctions.
Conditions where MMP-3 and MMP-13 have been implicated include tissue destruction such as in venous and diabetic ulcers, pressure sores, colon ulcers for example ulcerative colitis and Crohn""s disease, duodenal ulcers, and tissue destruction in arthritis, skin disorders such as dystrophic epidermolysis bulosa, dermatitis herpetiformis, or conditions caused by or complicated by embolic phenomena, such as chronic or acute cardiac or cerebral infarctions.
Another important function of certain MMPs is to activate other enzymes, including other MMPs, by cleaving the pro-domain from their protease domain. Thus, certain MMPs act to regulate the activities of other MMPs, so that over-production in one MMP may lead to excessive proteolysis of extracellular matrix by another.
Excessive production of MMP-3 is thought to be responsible for pathological tissue breakdown which underlies a number of diseases and conditions. For example, MMP-3 has been found in the synovium and cartilage of osteoarthritis and rheumatoid arthritis patients, thus implicating MMP-3 in the joint damage caused by these diseases. (See K. L. Sirum, C. E Brinkerhoff, Biochemistry, 1989, 28, 8691; Z. Gunja-Smith, H. Nagasse, J. F. Woessner, Biochem. J., 1989, 258, 115). MMP-13 is also thought to play an important role in the pathology of osteoarthritis and rheumatoid arthritis (M. Stahle-Backdahle, B. Sandstedt, K. Bruce, A. Lindahl, M. G. Jimenez, J. A. Vega, C. Lopez Otin, Lab. Invest., 1997, 76, 717-28; O. Lindy, Y. T. Konttinen, T. Sorsa, Y. Ding, S. Santavirta, A. Ceponis, C. Lopez Otin, Arthritis Rheum. 1997, 40, 1391-9).
Over-expression of MMP-3 is also thought to be responsible for much of the tissue damage and chronicity of chronic wounds, such as venous and diabetic ulcers, and pressure sores. (See M. Vaalamo, M. Weckroth, P. Puoakkainen, J. Kere, P. Saarinen, J. Lauharanta, U. K. Saarialho-Kere, Brit. J Dermatology, 1996, 135, 52-59).
During the healing of normal and chronic wounds, MMP-1 is expressed by migrating keratinocytes at the wound edges (U. K. Saarialho-Kere, S. O. Kovacs, A. P. Pentland, J Clin. Invest. 1993, 92, 2858-66). There is evidence which suggests MMP-1 is required for keratinocyte migration on a collagen type I matrix in vitro, and is completely inhibited by the presence of the non-selective MMP inhibitor SC44463 ((N4-hydroxy)-N1-[(1S)-2-(4-methoxyphenyl)methyl-1-((1R)-methylamino)carbonyl)]-(2R)-2-(2-methylpropyl)butanediamide) (B. K. Pilcher, J. A. Dumin, B. D. Sudbeck, S. M. Krane, H. G. Welgus, W.C. Parks, J. Cell Biol., 1997, 137, 1-13). Keratinocyte migration in vivo is essential for effective wound healing to occur.
MMP-2 and MMP-9 appear to play important roles in wound healing during the extended remodelling phase and the onset of re-epithelialisation, respectively (M. S. Agren, Brit. J. Dermatology, 1994, 131, 634-40; T. Salo, M. Mxc3xa4kxc3xa4nen, M. Kylmxc3xa4niemi, Lab. Invest., 1994, 70, 176-82). The potent, non-selective MMP inhibitor BB94 ((2S,3R)-5-methyl-3-{[(1S)-1-(methylcarbamoyl)-2-phenylethyl]carbamoyl}-2-[(2-thienylthio)methyl]hexanohydroxamic acid, batimastat), inhibits endothelial cell invasion of basement membrane, thereby inhibiting angiogenesis (G. Tarboletti, A. Garofalo, D. Belotti, T. Drudis, P. Borsotti, E. Scanziani, P. D. Brown, R. Giavazzi, J. Natl. Cancer Inst., 1995, 87, 293-8). There is evidence that this process requires active MMP-2 and/or 9.
Thus, non-selective MMP inhibitors which inhibit MMPs 1 and/or 2 and/or 9 would be expected to impair wound healing. As described above, MNP-14 is responsible for the activation of MMP-2, and thus inhibition of MMP-14 might also result in impaired wound healing.
The production of MMP-3 has also been thought to be involved in tissue damage in conditions where there is ulceration of the colon (as in ulcerative colitis and Crohn""s disease, see S. L. Pender, S. P. Tickle, A. J. Docherty, D. Howie, N. C. Wathen, T. T. MacDonald, J. Immunol., 1997, 158, 1582; C. J. Bailey, R. M. Hembry, A. Alexander, M. H. Irving, M. E. Grant, C. A. Shuttleworth, J. Clin. Pathol., 1994, 47, 113-6), or duodenum (see U. K. Saarialho-Kere, M. Vaalamo, P. Puolakkainen, K. Airola, W. C. Parks, M. L. Kaajalainen-Lindsberg, Am. J. Pathol., 1996, 148, 519-26). It is also likely that MMP-1 and MMP-2 are required during the healing phase of these conditions. A selective MMP-3 inhibitor would be more effective than a non-selective inhibitor.
MMP-3 has also been implicated in skin diseases such as dystrophic epidermolysis bullosa (T. Sato, K. Nomura, I. Hashimoto, Arch. Dermatol. Res., 1995, 287, 428) and dermatitis herpetiformis (K. Airola, M. Vaalamo, T. Reunala, U. K. Saarialho-Kere, J. Invest. Dermatology, 1995, 105, 184-9).
Rupture of atherosclerotic plaques by MMP-3 can lead to cardiac or cerebral infarction. (F. Mach, et al., Circulation, 1997, 96, 396-9.) Thus, MMP-3 inhibitors may find utility in the treatment of conditions caused by or complicated by embolic phenomena, such as chronic or acute cardiac or cerebral infarctions. MMP-12 (macrophage elastase) is thought to contribute to the pathology of atherosclerosis, gastro-intestinal ulcers and emphysema. For example, in a rabbit model of developing atherosclerosis, MMP-12 is expressed abundantly by macrophage foam cells (S. Matsumoto, et al, Am.J.Pathol. 1998, 153, 109). MMP-12 is also expressed abundantly by macrophages in the vicinity of shedding mucosal epithelium in human gastro-intestinal ulcers, such as those found in patients with ulcerative colitis and Crohn""s disease (M. Vaalamo, et al, Am.J.Pathol., 1998, 152, 1005). MMP-12 is thought to be important for the progression of lung damage by cigarette smoke. In a model of cigarette smoke-induced emphysema, mice lacking the gene for MMP-12 were resistant to developing the condition whereas wild-typemice suffered significant lung damage (R D Hautamaki, et al, Science, 1997, 277, 2002).
For recent reviews of MMPs, see Zask et al, Current Pharmaceutical Design, 1996, 2, 624-661; Beckett, Exp. Opin. Ther. Patents, 1996, 6, 1305-1315; and Beckett et al, Drug Discovery Today, vol 1(no.1), 1996, 16-26.
Alternative names for various MMPs and substrates acted on by these are shown in the table below (Zask et al, supra).
A number of publications, including some of those mentioned in the reviews above, describe compounds of the general formula shown below as MMP inhibitors. 
wherein xe2x80x9cAxe2x80x9d is known as the xe2x80x9calphaxe2x80x9d group and XCO is is a zinc-binding group such as a carboxylic acid or hydroxamic acid moiety.
The review by Beckett et al, mentioned above, states that a vast range of groups can be tolerated at the P2xe2x80x2 position without significantly affecting the behaviour of the compounds.
International Patent Application publication nos. WO96/33165 and WO96/33161 (British Biotech Pharmaceuticals Ltd.) generically disclose compounds of formula xe2x80x9cGENMMPxe2x80x9d above where, inter alia P1xe2x80x2 is optionally substituted phenyl(C1-C6)alkyl and P3xe2x80x2 is a group CHRxRy where Rx and Ry represent optionally substituted phenyl or heteroaryl rings. These compounds are said to be selective inhibitors of MMP-3 and MMP-7 relative to human fibroblast collagenase (MMP-1) and 72 KDa gelatinase (MMP-2).
International Patent Application publication no. WO96/16027 (Syntex Inc. and Agouron Pharmaceuticals Inc.) discloses MMP inhibitors of the general formula xe2x80x9cGENMMPxe2x80x9d as shown above wherein COX includes CO2H and CONHOH, P1xe2x80x2 is a group R2X which includes optionally substituted aryl(C0-4 alkylene), and P3xe2x80x2 is (CH2)pR7 where p is 0 to 4, provided that when p is not 0 then R2X is biphenylalkyl, and R7 is aryl or heteroaryl. Compounds where p is 0, 2 or 3 are said to be preferred, and compounds where p is 0 and COX is CO2H or CONHOH are preferred for matrilysin (i.e. MMP-7) inhibition.
A number of compounds from said publication WO96/16027 of general formula xe2x80x9cGENMMPxe2x80x9d have been disclosed by the Agouron group (2nd Winter Conference on Medicinal and Bioorganic Chemistry, Steamboat Springs, Colo., USA, January 1997) e.g. wherein X is OH, P1xe2x80x2 is an optionally 4xe2x80x2-substituted biarylpropyl group, P2xe2x80x2 is isobutyl and P3xe2x80x2 is 4-methoxycarbonylphenyl. Such compounds were reported to have poor to moderate MMP-3/MMP-2 selectivity. It was found that use of an alpha-substituent and replacement of the 4-methoxycarbonylphenyl moiety with a 4-methylthiophenyl group greatly enhanced the MMP-3/MMP-2 selectivity.
The review by Beckett (above) also mentions an Agouron compound (#31 of the review) of general formula xe2x80x9cGENMMPxe2x80x9d wherein X is OH, P1xe2x80x2 is biphenylpropyl, P2xe2x80x2 is t-butyl, and P3xe2x80x2 is 4-pyridyl. This compound has almost identical Ki values for MMP-3 and MMP-2, so is not selective for MMP-3 over MMP-2.
International Patent Application no. WO95/12603 (Syntex) discloses compounds, said to be MMP-3 and MMP-7 inhibitors, of formula xe2x80x9cGENMMPxe2x80x9d above, where P3xe2x80x2 is a substituted phenyl moiety and P1xe2x80x2 includes arylalkyl.
We have now found a group of MMP inhibitor compounds with good activity for MMP-3, MMP-12 and MMP-13, and good selectivity for MMP-3 over other MMPs such as MMPs-1, 2, 9 and 14. It has been found, for this group of compounds, that the MMP-3 selectivity is remarkably dependent on a particular combination of P1xe2x80x2 and P3xe2x80x2 substituents, which effect could not have been predicted from the art mentioned above.
Thus, according to the present invention, there are provided compounds of formula (I): 
and pharmaceutically acceptable salts thereof, wherein
R1 is H, OH, C1-4 alkyl, C1-4 alkoxy, or C2-4 alkenyl,
R2 is C1-6 alkyl optionally substituted by fluoro, indolyl, imidazolyl, SO2(C1-4 alkyl), C5-7 cycloalkyl,
or by an optionally protected OH, SH, CONH2, CO2H, NH2 or NHC(xe2x95x90NH)NH2 group, C5-7 cycloalkyl optionally substituted by C1-6 alkyl,
or is benzyl optionally substituted by optionally protected OH, C1-6 alkoxy, benzyloxy or benzylthio,
wherein the optional protecting groups for said OH, SH, CONH2, NH2 and NHC(xe2x95x90NH)NH2 groups are selected from C1-6 alkyl, benzyl, C1-6 alkanoyl,
and where the optional protecting groups for said CO2H is selected from C1-6 alkyl or benzyl,
R3, R5 and R6 are each independently selected from H and F,
R4 is CH3, Cl or F,
X is HO or HONH,
Y is a direct link or O.
Z is either a group of formula (a): 
where R10 is C1-4 alkyl, C1-4 alkoxymethyl, hydroxy(C2-4 alkyl), carboxy(C1-4 alkyl) or (amino or dimethylamino)C2-4 alkyl,
and R11 is phenyl, naphthyl or pyridyl optionally substituted by up to three substituents independently selected from halo and methyl;
or (b) 
R14 is H, OH, CH3 or halo,
Ar is a group of formula (c), (d) or (e): 
wherein
A is N or CR12,
B is N or CR13,
provided that A and B are not both N,
R7 and R9 are each independently H or F,
R8, R12 and R13 are each independently H, CN, C1-6 alkyl, hydroxy(C1-6 alkyl), hydroxy(C1-6)alkoxy, C1-6 alkoxy(C1-6)alkoxy,(amino or dimethylamino)C1-6 allyl, CONH2, OH, halo, C1-6 alkoxy, (C1-6 alkoxy)methyl, piperazinylcarbonyl, piperidinyl, C(NH2)xe2x95x90NOH or C(xe2x95x90NH)NHOH, with the proviso that at least two of R8, R12 and R13 are H.
xe2x80x9cAlkylxe2x80x9d groups, including the alkyl moiety of xe2x80x9calkoxyxe2x80x9d groups, and xe2x80x9calkenylxe2x80x9d groups, can be straight or branched where the number of carbon atoms allows.
xe2x80x9cHaloxe2x80x9d means F, Cl, Br or I.
The compounds of the present invention are MMP inhibitors, and are especially potent and selective MMP-3 inhibitors, especially with good selectivity over MMPs-1,2,9 and/or 14. In addition, certain of the compounds of the invention have useful MMP-12 and/or MMP-13 inhibitory activity.
Preferably, R1 is H, OH, C1-4 alkyl or C1-4alkoxy.
More preferably, R1 is H, OH, n-propyl or ethoxy.
Most preferably R1 is H.
Preferably R2 is C1-6 alkyl optionally substituted by indolyl, C1-6 alkylthio, SO2(C1-4alkyl), C5-7 cycloalkyl, OH or SH, C5-7 cycloalkyl optionally substituted by C1-6 alkyl, or R2 is benzyl.
More preferably R2 is C1-6 alkyl optionally substituted by OH, SO2(C1-4 alkyl) or C5-7 cycloalkyl, cyclohexyl optionally substituted by C1-6 alkyl, or R2 is benzyl.
Yet more preferably, R2 is cyclohexylmethyl, isopropyl, 1-methylcyclohexyl, t-butyl, C(CH3)2SO2CH3, benzyl or C(CH3)2OH.
Further yet more preferably R2 is isopropyl, t-butyl or benzyl.
Most preferably, R2 is t-butyl.
Preferably, Z is a group of formula (a): 
where R10 is C1-4 alkyl, C1-4 alkoxymethyl or hydroxy(C2-4 alkyl) and R11 is phenyl or pyridyl, optionally substituted by up to three substituents independently selected from halo and methyl,
or Z is 
More preferably Z is a group of formula (a): 
where R10 is C1-4 alkyl, C1-4 alkoxymethyl or hydroxy(C2-4 alkyl), and R11 is phenyl, pyridin-4-yl or pyridin-3-yl,
or Z is 
Yet more preferably Z is a group of formula (a): 
where R10 is CH3, CH2OCH3, or CH2OH, and R11 is phenyl, pyridin-4-yl or pyridin-3-yl, or Z is 
Most preferably, Z is a group of formula 
Preferably R3 is H.
Preferably R4 is F when Y is O.
Preferably R4 is Cl or CH3 when Y is a direct link.
Preferably R5 is H.
Preferably R6 is H.
Preferably Ar is a group of formula (c), 
wherein
A is CR12 and B is CR13,
R7 and R9 are each independently H or F,
R8 and R13 are each independently H, F, Cl, CN, CONH2, CH3 or OCH3, and
R12 is H, C1-6 alkyl, CN, hydroxy(C2-6 alkyl), (amino or dimethylamino)C2-6 alkyl, CONH2, OH, halo, C1-6 alkoxy, (C1-6 alkoxy)methyl, piperazinylcarbonyl, piperidinyl, C(NH2)NOH or C(xe2x95x90NH)NHOH.
More preferably Ar is a group of formula (c), 
wherein A is CR12, B is CR13, and R7, R8 and R9 are H.
Yet more preferably, Ar is a group of formula (c), 
wherein
A is CR12, B is CR13, R7, R8 and R9 are H,
R12 is H, C1-6 alkyl, CN, hydroxy(C2-6 alkyl), (amino or dimethylamino)C2-6 alkyl, CONH2, OH, halo, C1-6 alkoxy, (C1-6 alkoxy)methyl, C(NH2)xe2x95x90NOH or C(xe2x95x90NH)NHOH, and R13 is H, OCH3, CN, CONH2, CH3 or F.
Further yet more preferably Ar is phenyl, 3-methoxyphenyl, 4-cyanophenyl, 3-cyanophenyl, 3-carbamoylphenyl or 4-hydroxyamidinophenyl. Most preferably, Ar is phenyl or 3-methoxyphenyl.
A preferred group of substances are those where the substituents have the values mentioned in the Examples, viz.:
R1 is H, OH, n-propyl or ethoxy,
R2 is t-butyl, isopropyl or benzyl,
Z is a group of formula 
where R10 is CH3, CH2OCH3, or CH2OH, and R11 is phenyl, pyridin-4-yl or pyridin-3-yl,
or Z is 
R3 is H,
R4 is CH3, Cl or F,
R5 is H,
R6 is H,
and Ar is phenyl, 3-methoxyphenyl, 4-cyanophenyl, 3-cyanophenyl, 3-carbamoylphenyl or 4-hydroxyamidinophenyl, and the salts thereof.
Another preferred group are the compounds are those of the Examples below, and the salts thereof.
The most preferred substances are selected from Examples 3, 4, 8, 14, 15, 16, 22, 29, 30, 31 and 32 below, and the salts thereof.
Pharmaceutically-acceptable salts are well known to those skilled in the art, and for example include those mentioned in the art cited above, and by Berge et al, in J.Pharm.Sci., 66, 1-19 (1977). Suitable acid addition salts are formed from acids which form non-toxic salts and include the hydrochloride, hydrobromide, hydroiodide, nitrate, sulphate, bisulphate, phosphate, hydrogenphosphate, acetate, trifluoroacetate, gluconate, lactate, salicylate, citrate, tartrate, ascorbate, succinate, maleate, fumarate, gluconate, formate, benzoate, methanesulphonate, ethanesulphonate, benzenesulphonate and p-toluenesulphonate salts.
Pharmaceutically acceptable base addition salts are well known to those skilled in the art, and for example include those mentioned in the art cited above, and can be formed from bases which form non-toxic salts and include the aluminium, calcium, lithium, magnesium, potassium, sodium and zinc salts, and salts of non-toxic amines such as diethanolamnine.
Certain of the compounds of the formula (I) may exist as geometric isomers. The compounds of the formula (I) may possess one or more asymmetric centres, apart from the specified centres in formula (I), and so exist in two or more stereoisomeric forms. The present invention includes all the individual stereoisomers and geometric isomers of the compounds of formula (I), apart from the specified centres in formula (I), including the group Z, and mixtures thereof.
Another aspect of the invention is a pharmaceutical composition comprising a compound or salt according to the above definitions and a pharmaceutically-acceptable adjuvant, diluent or carrier.
Yet another aspect of the invention is a compound or salt according to the above definitions for use as a medicament.
A further aspect of the invention is the use of a compound or salt according to the above definitions for the manufacture of a medicament for the treatment of a condition mediated by one or more matrix metalloproteases, especially MMP-3 and/or MMP-12 and/or MMP-13.
Yet another aspect of the invention is a method of treatment of a condition mediated by one or more matrix metalloproteases, especially MMP-3 and/or MMP-12 and/or MMP-13.
It is to be appreciated that reference to treatment includes prophylaxis as well as the alleviation of established symptoms of MMP-mediated conditions.
The invention further provides Methods for the production of compounds of the invention, which are described below and in the Examples. The skilled man will appreciate that the compounds of the invention could be made by methods other than those specifically described herein, by adaptation of the methods herein described in the sections below and/or adaptation thereof, and of methods known in the art. Suitable guides to synthesis, functional group transformations, use of protecting groups, etc. are, for example, xe2x80x9cComprehensive Organic Transformationsxe2x80x9d by R C Larock, VCH Publishers Inc. (1989), xe2x80x9cAdvanced Organic Chemistryxe2x80x9d by J March, Wiley Interscience (1985), xe2x80x9cDesigning Organic Synthesisxe2x80x9d by S Warren, Wiley Interscience (1978), xe2x80x9cOrganic Synthesisxe2x80x94The Disconnection Approachxe2x80x9d by S Warren, Wiley Interscience (1982), xe2x80x9cGuidebook to Organic Synthesisxe2x80x9d by R K Mackie and D M Smith, Longman (1982), xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d by T W Greene and PGM Wuts, John Wiley and Sons Inc. (1991), and P J Kocienski, in xe2x80x9cProtecting Groupsxe2x80x9d, Georg Thieme Verlag (1994).
In the Methods below, unless otherwise specified, the substituents are as defined above with reference to the compounds of formula (I) above.
Method 1
Compounds of formula (I) where X is OH can be obtained via the corresponding compound of formula (II) below where X, is a group capable of being transformed to a carboxy group in conditions which do not result in substantial transformation of the other parts of the compound (II). A suitable example of such a group is CO2(t-butyl or methyl). The t-butyl group can be cleaved by reaction with an acid such as hydrogen chloride or trifluoroacetic acid (TFA) in a suitable solvent such as anhydrous dichloromethane or dioxane, at a suitable temperature such as between 0xc2x0 C. and 20xc2x0 C. The methyl ester can be hydrolysed by a hydroxide such as lithium hydroxide, in a suitable solvent system such as a tetrahydrofuran/water mixture, suitably at room temperature. 
Compounds of formula (II) may be made by methods known in the art, and exemplified in the Preparations below, for example by coupling of suitable amine and acid derivatives, which are available by known chemistry.
Method 2
Compounds of formula (I) where X is NHOH can be obtained from the corresponding compounds of formula (I) where X is OH via treatment of the compounds of formula (I) where X is OH with hydroxylamine, such as by generation from a hydroxylamine salt such as the hydrochloride, by a suitable base such as a tertiary amine, e.g. diisopropylethylamine, in a suitable solvent such as N,N-dimethylformamide (DMF), and a coupling agent such as N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethaninium hexafluorophosphate N-oxide (xe2x80x9cHATUxe2x80x9d, which reagent is described in Tet.Letts. (1994) 35, 2279), at a suitable temperature such as from 0-20xc2x0 C.
Compounds of formula (I) where X is OH may be made by conventional methods and the methods described herein.
Method 3
Compounds of formula (I) where X is NHOH can be obtained from the corresponding compounds of formula (I) where X is OH via treatment of the compounds of formula (I) where X is OH with O-allylhydroxylamine, such as by generation from a O-allylhydroxylamine salt such as the hydrochloride, by a suitable base such as a tertiary amine e.g. diisopropylethylamine, in a suitable solvent such as N,N-dimethylformamide (DMF) or dichloromethane, and a coupling agent, e.g. a peptide coupling agent such as 7-azabenzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate (xe2x80x9cPyAOPxe2x80x9d), at a suitable temperature such as from 0-20xc2x0 C. This stage gives compounds of formula (III) below.
This coupling method is generally described in Tet.Letts.(1994) 35,2279. 
Compounds of formula (III) can be transformed into compounds of formula (I) where X is NHOH by treatment with ammonium formate in the presence of a suitable catalyst such as bis(triphenylphosphine)palladium(II) acetate, in a suitable solvent such as aqueous ethanol, at a suitable temperature such as the refluxing temperature of aqueous ethanol.
Method 4
Compounds of formula (I) where X is NHOH and R1 is OH can be made via reaction of a compound of formula (IV) with hydroxylamine, for example via generation from a hydroxylamine salt such as the hydrochloride with a suitable base such as sodium methoxide in a suitable solvent such as methanol. 
Compounds of formula (IV) may be made by conventional methods as are exempllified in the Preparations below.
Method 5
Compounds of formula (I) may be made from compounds of formula (V) below via cross-coupling with a compound of formula (VI) below: 
where X2 is a protected acid such as the t-butyl or methyl ester group and LG is a cross-coupling leaving group such as I, Br or OSO2CF3. The cross-coupling reaction can be carried out in the presence of a catalyst such as bis(tri-o-tolyl)phosphine palladium(II) acetate with a suitable base such as triethylamine, in a suitable solvent such as acetonitrile or DMF, at a suitable temperature such as between 50-150xc2x0 C. This type of reaction is generally described by Heck in Tet.Letts. (1984) 25, 2271, and numerous other articles.
Compounds of the formula (V) can be made by conventional methods such as by adaptation of those described in the Preparations below.
Compounds of the formula (VI) can be made by conventional methods such as by adaptation of those described in the Preparations below, and by reference to the following articles: Synthesis (1984) 709; J Chem Soc Perkin Trans I(1977) 1841; J Org Chem (1994) 59, 6095; ibid, (1979) 44, 4444 and Tet.Letts.(1997) 38, 1749.
The resultant product from this reaction is a mixture of compounds of formula (VIIa) and (VIIb) wherein X2 is defined as above for compounds of formula (V). 
The compounds of formula (VIIa) and (VIIb) can be transformed into a compound of formula (I) where X is OH, by reduction of the olefinic bond using conventional methods, such as hydrogenation in the presence of a catalyst, or by reaction with diimide, which may be generated, for example, from p-toluenesulphonyl hydrazide, and deprotection of the protected acid moiety X2.
It will be apparent to those skilled in the art that other protection and subsequent deprotection regimes during synthesis of a compound of the invention may be achieved by conventional techniques, for example as described in the volumes by Greene and Wuts, and Kocienski, supra.
Where desired or necessary the compound of formula (I) is converted into a pharmaceutically acceptable salt thereof. A pharmaceutically acceptable salt of a compound of formula (I) may be conveniently be prepared by mixing together solutions of a compound of formula (I) and the desired acid or base, as appropriate. The salt may be precipitated from solution and collected by filtration, or may be collected by other means such as by evaporation of the solvent.
Certain compounds of the invention may be interconverted into certain other compounds of the invention by well-known methods from the literature.
Compounds of the invention are available by either the methods described herein in the Methods and Examples or suitable adaptation thereof using methods known in the art. It is to be understood that the synthetic transformation methods mentioned herein may be carried out in various different sequences in order that the desired compounds can be efficiently assembled. The skilled chemist will exercise his judgement and skill as to the most efficient sequence of reactions for synthesis of a given target compound.
The compounds and salts of the invention may be separated and purified by conventional methods.
Separation of diastereomers may be achieved by conventional techniques, e.g. by fractional crystallisation, chromatography or H.P.L.C. of a stereoisomeric mixture of a compound of formula (I) or a suitable salt or derivative thereof. An individual enantiomer of a compound of formula (I) may also be prepared from a corresponding optically pure intermediate or by resolution, such as by H.P.L.C. of the corresponding racemate using a suitable chiral support or by fractional crystallisation of the diastereomeric salts formed by reaction of the corresponding racemate with a suitably optically active acid or base.
For human use, the compounds of formula (I) or their salts can be administered alone, but will generally be administered in admixture with a pharmaceutically acceptable diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice. For example, they can be administered orally, including sublingually, in the form of tablets containing such excipients as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents. The compound or salt could be incorporated into capsules or tablets for targetting the colon or duodenum via delayed dissolution of said capsules or tablets for a particular time following oral administration. Dissolution could be controlled by susceptibility of the formulation to bacteria found in the duodenum or colon, so that no substantial dissolution takes places before reaching the target area of the gastrointestinal tract. The compounds or salts can be injected parenterally, for example, intravenously, intramuscularly or subcutaneously. For parenteral administration, they are best used in the form of a sterile aqueous solution or suspension which may contain other substances, for example, enough salt or glucose to make the solution isotonic with blood. They can be administered topically, in the form of sterile creams, gels, suspensions, lotions, ointments, dusting powders, sprays, drug-incorporated dressings or via a skin patch. For example they can be incorporated into a cream consisting of an aqueous or oily emulsion of polyethylene glycols or liquid paraffin, or they can be incorporated into an ointment consisting of a white wax soft paraffin base, or as hydrogel with cellulose or polyacrylate derivatives or other viscosity modifiers, or as a dry powder or liquid spray or aerosol with butane/propane, HFA or CFC propellants, or as a drug-incorporated dressing either as a tulle dressing, with white soft paraffin or polyethylene glycols impregnated gauze dressings or with hydrogel, hydrocolloid, alginate or film dressings. The compound or salt could also be administered intraocularly as an eye drop with appropriate buffers, viscosity modifiers (e.g. cellulose derivatives), preservatives (e.g. benzalkonium chloride (BZK)) and agents to adjust tenicity (e.g. sodium chloride). Such formulation techniques are well-known in the art.
All such formulations may also contain appropriate stabilisers and preservatives.
For oral and parenteral administration to human patients, the daily dosage level of the compounds of formula (I) or their salts will be from 0.001 to 20, preferably from 0.01 to 20, more preferably from 0.1 to 10, and most preferably from 0.5 to 5 mg/kg (in single or divided doses). Thus tablets or capsules of the compounds will contain from 0.1 to 500, preferably from 50 to 200, mg of active compound for administration singly or two or more at a time as appropriate.
For topical administration to human patients with chronic wounds, the daily dosage level of the compounds, in suspension or other formulation, could be from 0.01 to 50mg/ml, preferably from 0.3 to 30 mg/ml.
The physician in any event will determine the actual dosage which will be most suitable for a an individual patient and it will vary with the age, weight and response of the particular patient. The above dosages are exemplary of the average case; there can of course be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
Test Methods
The ability of compounds to inhibit the cleavage of fluorogenic peptides by MMPs 1, 2, 3, 9 12, 13 and 14 is described below.
The assays for MMPs 2, 3, 9, and 14 are based upon the original protocol described by Knight et al. (Fed.Euro.Biochem.Soc., 296 (3), 263-266; 1992) with the slight modifications given below.
Inhibition of MMP-1
(i) Enzyme Preparation
Catalytic domain MMP-1 was prepared at Pfizer Central Research. A stock solution of MMP-1 (1 xcexcM) was activated by the addition of aminophenylmercuric acetate (APMA), at a final concentration of 1 mM, for 20 minutes at 37xc2x0 C. MMP-1 was then diluted in Tris-HCl assay buffer (50 mM Tris, 200 mM NaCl, 5 mM CaCl2, 20 xcexcM ZnSO4, 0.05% Brij 35) pH 7.5 to a concentration of 10 nM. The final concentration of enzyme used in the assay was 1 nM.
(ii) Substrate
The fluorogenic substrate used in this assay was Dnp-Pro-xcex2-cyclohexyl-Ala-Gly-Cys(Me)-His-Ala-Lys(N-Me-Ala)-NH2 as originally described by Bickett et al (Anal. Biochem, 212, 58-64, 1993). The final substrate concentration used in the assay was 10 xcexcM.
(iii) Determination of Enzyme Inhibition
Test compounds were dissolved in dimethyl sulphoxide and diluted with assay buffer so that no more than 1% dimethyl sulphoxide was present. Test compound and enzyme were added to each well of a 96 well plate and allowed to equilibrate for 15 minutes at 37xc2x0 C. in an orbital shaker prior to the addition of substrate. Plates were then incubated for 1 hour at 37xc2x0 C. prior to determination of fluorescence (substrate cleavage) using a fluorimeter (Fluostar; BMG LabTechnologies, Aylesbury, UK) at an excitation wavelength of 355 nm and emission wavelength of 440 nm. The potency of inhibitors was measured from the amount of substrate cleavage obtained using a range of test compound concentrations, and, from the resulting dose-response curve, an IC50 value (the concentration of inhibitor required to inhibit 50% of the enzyme activity) was calculated.
Inhibition of MMP-2, MMP-3 and MMP-9
(i) Enzyme Preparation
Catalytic domain MMP-2, MMP-3 and MMP-9 were prepared at Pfizer Central Research. A stock solution of MMP-2, MMP-3 or MMP-9 (1 xcexcM) was activated by the addition of aminophenylmercuric acetate (APMA). For MMP-2 and MMP-9, a final concentration of 1 mM APMA was added, followed by incubation for 1 hour at 37xc2x0 C. MMP-3 was activated by the addition of 2 mM APMA, followed by incubation for 3 hours at 37xc2x0 C. The enzymes were then diluted in Tris-HCl assay buffer (100 mM Tris, 100 mM NaCl, 10 mM CaCl2 and 0.16% Brij 35, pH 7.5), to a concentration of 10 nM. The final concentration of enzyme used in the assays was 1 nM.
(ii) Substrate
The fluorogenic substrate used in this screen was Mca-Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met-Lys(Dnp)-NH2 (Bachem Ltd, Essex, UK) as originally described by Nagase et al (J.Biol.Chem., 269(33), 20952-20957, 1994). This substrate was selected because it has a balanced hydrolysis rate against MMPs 2, 3 and 9 (kcat/km of 54,000, 59,400 and 55,300 sxe2x88x921 Mxe2x88x921 respectively). The final substrate concentration used in the assay was 5 xcexcM.
(iii) Determination of Enzyme Inhibition
Test compounds were dissolved in dimethyl sulphoxide and diluted with test buffer solution (as above) so that no more than 1% dimethyl sulphoxide was present. Test compound and enzyme were added to each well of a 96 well plate and allowed to equilibrate for 15 minutes at 37xc2x0 C. in an orbital shaker prior to the addition of substrate. Plates were then incubated for 1 hour at 37xc2x0 C. prior to determination of fluorescence using a fluorimeter (Fluostar; BMG LabTechnologies, Aylesbury, UK) at an excitation wavelength of 328 nm and emission wavelength of 393 nm. The potency of inhibitors was measured from the amount of substrate cleavage obtained using a range of test compound concentrations, and, from the resulting dose-response curve, an IC50 value (the concentration of inhibitor required to inhibit 50% of the enzyme activity) was calculated.
Inhibition of MMP-12 (Human Macrophage Elastase)
(i) Enzyme Preparation
Catalytic domain MMP-12 (200 xcexcg/ml) was used. MMP-12 was diluted in Tris-HCl assay buffer (50 mM Tris, 200 mM NaCl, 5 mM CaCl2, 20 xcexcM ZnSO4, 0.02% Brij 35) pH 7.4 to 240 ng/ml. The final concentration of enzyme used in the assay was 60 ng/ml.
(ii) Substrate
The fluorogenic substrate used in this assay was DNP-Pro-Cha-Gly-Cys(Me)-His-Ala-Lys(NMA)-NH2. The final substrate concentration used in the assay was 10 xcexcM.
(iii) Determination of Enzyme Inhibition
Test compounds were dissolved in dimethyl sulphoxide and diluted with assay buffer so that no more than 1% dimethyl sulphoxide was present. Test compound and enzyme were added to each well of a 96 well plate and allowed to equilibrate for 15 minutes at room temperature in an orbital shaker prior to the addition of substrate. Plates were then incubated for 2 hours at room temperature prior to the determination of fluorescence (substrate cleavage) using a fluorimeter at an excitation wavelength of 360 nm and emission wavelength of 460 nm. The potency of inhibitors was measured from the amount of substrate cleavage obtained using a range of test compound concentrations, and, from the resulting dose-response curve, an IC50 value (the concentration of inhibitor required to inhibit 50% of the enzyme activity) was calculated.
Inhibition of MMP-13
(i) Enzyme Preparation
Human recombinant MMP-13 was prepared by PanVera Corporation (Madison, Wis.) and characterized at Pfizer (Groton, Conn.). A 1.9 mg/ml stock was activated with 2 mM APMA for 2 hours at 37xc2x0 C. MMP-13 was then diluted in assay buffer (50 mM Tris, 200 mM NaCl, 5 mM CaCl2, 20 xcexcM ZnCl2 and 0.02% Brij 35) at pH 7.5 to a concentration of 5.3 nM. The final concentration of enzyme used in the assay was 1.3 nM.
(ii) Substrate.
The fluorogenic substrate used in this screen was Dnp-Pro-Cha-Gly-Cys(Me)-His-Ala-Lys(NMA)-NH2. The final substrate concentration used in the assay was 10 xcexcM.
(iii) Determination of Enzyme Inhibition
Test compounds were dissolved in dimethyl sulphoxide and diluted with assay buffer so that no more than 1% dimethyl sulphoxide was present. Test compound and enzyme were added to each well of a 96 well plate. The addition of substrate to each well initiated the reaction. Fluorescence intensity was determined on a 96 well plate fluorometer (Cytofluor II; PerSeptive Biosystems, Inc, Framingham, Mass.) at an excitation wavelength of 360 nm and emission wavelength of 460 nm. The potency of inhibitors was measured from the amount of substrate cleavage obtained using a range of test compound concentrations, and, from the resulting dose-response curve, an IC50 value (the concentration of inhibitor required to inhibit 50% of the enzyme activity) was calculated.
Inhibition of MMP-14
(i) Enzyme Preparation
Catalytic domain MMP-14 was purchased from Prof Tschesche, Department of Biochemistry, Faculty of Chemistry, University of Bielefeld, Germany. A 10 xcexcM enzyme stock solution was activated for 20 minutes at 25xc2x0 C. following the addition of 5 xcexcg/ml of trypsin (Sigma, Dorset, UK). The trypsin activity was then neutralised by the addition of 50 xcexcg/ml of soyabean trypsin inhibitor (Sigma, Dorset, UK), prior to dilution of this enzyme stock solution in Tris-HCl assay buffer (100 mM Tris, 100 mM NaCl, 10 mM CaCl2 and 0.16% Brij 35, pH 7.5) to a concentration of 10 nM. The final concentration of enzyme used in the assay was 1 nM.
(ii) Substrate
The fluorogenic substrate used in this screen was Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 (Bachem Ltd, Essex, UK) as described by Will et al (J.Biol.Chem., 271(29), 17119-17123, 1996). The final substrate concentration used in the assay was 10 xcexcM.
Determination of enzyme inhibition by test compounds was performed as described for MMPs 2, 3 and 9.
Some activity data for certain compounds of the Examples are presented in the Table below.
The compounds of Examples 3, 4, 8, 14, 15, 16, 22,29, 30, 31 and 32 had MMP-3/MMP-2 selectivities in the range 195-930.